The optimal duration of anti-PD-1 for cancer therapy has not been tested, especially when using combination therapy. Epidermal growth factor receptor (EGFR) pathway blocker was the top compound that enhanced T-cell killing of tumor cells in a high-throughput immune-oncology screen, possibly by stimulate the antigen presentation machinery and other mechanisms. We explored the effect of combination of EGFR inhibition with a short course of anti-PD-1 therapy in patients with recurrent or metastatic head and neck squamous cell carcinoma (R/M HNSCC).We analyzed the effect of a short course of anti-PD-1 with continuous afatinib on the survival of a real-world cohort of R/M HNSCC patients. Patient characteristics, treatments, efficacies, and toxicities were reviewed and recorded for analysis.From November 2016 to May 2018, 51 consecutive patients received pembrolizumab and afatinib. The cutoff date was June 30, 2022. The most common toxicities  were diarrhea (62.7%), skin rash (43.1%), mucositis (31.4%), and paronychia (23.5%). The objective response rate was 54.9% . Median progression-free survival was 5.9 months (95% CI: 4.4–7.6 months), and the median overall survival was 10.5 months (95% CI: 6.8–16.5 months). The 12-month, 24-month, 36-month, and 48-month survival rate was 47.0%, 22.5%, 17.7%, and 12.6% respectively.This retrospective study showed that short course pembrolizumab with afatinib therapy has acceptable efficacy in R/M HNSCC patients. The durable response and long-term survival rates were similar to prospective clinical trials. Short course anti-PD-1 therapy, especially in combination with EGFR blocker, is worth for further prospective study. Anti-programmed death-1 (anti-PD-1) antibodies, including pembrolizumab  and nivoAnti-PD-1 combination therapy has been used for overcoming intrinsic resistance and improving treatment efficacy in HNSCC. Several phase II studies have shown the effectiveness of epidermal growth factor receptor (EGFR) pathway inhibition for improving anti-PD-1 efficacy in HNSCC patients. Two studies that used cetuximab with pembrolizumab  or nivolCombing with that best partner to enhance T-cell killing of tumor cells, a shorter course anti-PD-1 therapy may be feasible. We retrospectively reviewed real-world data from a medical center in Taiwan to explore the efficacy of a short course of pembrolizumab combined with continuous afatinib for HNSCC patients.The medical record retrospective review focusing on recurrent or metastatic HNSCC patients taking anti-PD-1 was approved by the institutional review board (IRB) of the National Taiwan University Hospital (NTUH), Taipei, Taiwan . Informed consent has been waived by the IRB of NTUH, Taipei, Taiwan, as this was a retrospective study. All methods were performed in accordance with the relevant guidelines and regulations.Patients with head and neck cancer fulfilling the following criteria were enrolled for analysis: (1) documented HNSCC ; (2) recurrent or metastatic disease not suitable for further curative treatment; (3) no other concurrent cancers; and (4) taking a combination of pembrolizumab and afatinib for palliation. After obtaining positive results in a phase II trial , we focuThe study analyzed the following characteristics: age, the curative treatment plan, and history of chemotherapy or anti-cancer therapy. Patients' history of alcohol drinking, betel nut chewing, and cigarette smoking were also recorded. Definitive therapy included surgical tumor excision, radiotherapy alone, or chemoradiation. Time from definitive therapy to recurrence or metastases was defined from the last date of definitive therapy to the first date of known recurrence or metastases.The medical records and laboratory data were reviewed for toxicity analysis. Severity was evaluated by certified medical oncologists and was recorded regularly in the electronic health record.Tumor response was evaluated using contrast imaging (magnetic resonance imaging or computed tomography). In the case of patients who did not have a contrast image evaluation, data on tumor size measured with a ruler were used in the analysis. RECIST 1.1  was usedWe reviewed medical records as a reference for survival status. Progression-free survival (PFS) was defined as the period from the time of first taking pembrolizumab to the time of disease progression or death. Overall survival (OS) was defined as the period from the time of first taking pembrolizumab to the time of death. For responders defined by RECIST 1.1 , the durThe Kaplan–Meier procedure was used for analysis of survival. The Cox proportional hazards regression model was used for multivariate analysis. The log-rank test was used to compare survival. MedCalc (Version 20.111) was used for the analyses.From Nov 2016 to Sep 2018, 51 consecutive HNSCC patients fulfilling the reviewing criteria were included in the analysis (Table The majority of patients (67%) took four cycles of pembrolizumab, and 52% of patients took a 200-mg fixed dose of pembrolizumab every three weeks. All patients took 40 mg of afatinib daily as the initial dose of treatment. The prescription of afatinib was maintained until disease progression, intolerable toxicity, or the physician's decision to discontinue. The median treatment duration for afatinib was 5.9 months (range: 0.5 to 25.8 months).For patients taking pembrolizumab and afatinib, the most common all-grade toxicities Table  were diaIn this cohort, 47 patients had contrast images for disease evaluation, while two patients were given a physical examination. The tumors were measured by rulers, and the size of tumor was recorded in the medical record. One patient was diagnosed with clinical progression. One patient did not have evaluable disease. In the case of the 47 patients with contrast images for evaluation, the median number of examinations of the contrast images was 3 range: 2–12). The median interval of contrast image evaluation was 3.1 months (range: 0.5–20.4 months). The objective response rate (ORR) was 54.9%   and OS .We included the history of alcohol drinking, cigarette smoking, betel nut chewing, cetuximab use, cancer types , time from definitive treatment to recurrence, and pembrolizumab dose (200 mg vs. 2 mg/kg) in the Cox proportional hazards regression analysis of OS. No difference was observed in this analysis Table . There wThis is a retrospective analysis of real-world data for patients using limited cycles of pembrolizumab with continuous afatinib for the treatment of HNSCC. The median cycles of pembrolizumab treatment are four cycles, approximately before the first image evaluation. It was an appropriate time for discussing further treatment plans with the patients and their family. In this retrospective analysis, the reasons for holding pembrolizumab treatment are not written down in medical records. However, financial restraint could be the most possible reason to hold the therapy. In this study, the treatment of short-course pembrolizumab plus continuous afatinib still showed a comparative clinical effect. The ORR of recurrent and/or metastatic HNSCC patients to the treatment was 54.9%. The median PFS was 5.9 months, and the median OS was 10.5 months. The toxicities were tolerable and manageable. The response rate was comparable to that in the prospective phase II study using afatinib–pembrolizumab for HNSCC . The lonIn addition to regulating tumor growth , the EGFThe high costs of anti-PD-1 and reimbursement constraints limit patients' access to anti-PD-1 therapy . In TaiwThe duration of anti-PD-1 treatment is also of interest. In metastatic melanoma patients, four cycles of the CTLA-4 antibody ipilimumab demonstrated a durable response . The surThe report also identified several challenges that require further investigation. A key issue in TKI and anti-PD-1 combination therapy is the management of TKI associated toxicities. In this cohort, 70.6% of patients required dose reductions. In other clinical trials using afatinib-pembrolizumab , lenvatiThis study has several limitations. First, it is a retrospective study. Efficacy might have been overestimated and toxicities underestimated. The heterogeneous characteristics in our study also limited further subgroup analysis for predicting treatment benefits. Second, the cost of these treatment was not reimbursed in Taiwan in this retrospective study period. Patients who can afford the cost of therapy may reflect a better family support and financial status. The prognosis of these patients could be better in this cohort. Lastly, PD-L1 expression serves as a biomarker for predicting clinical response to anti-PD-1 therapy. However, PD-L1 testing is not widely available in Taiwan at this time. As a result, it may be difficult to determine the correlation between PD-L1 expression and clinical response rate. Our prospective study found that higher PD-L1 expression was associated with better outcomes in patients with HNSCC receiving afatinib-pembrolizumab .In conclusion, this retrospective analysis of real-world data showed acceptable efficacy of short-course pembrolizumab with continuous afatinib against HNSCC. The substantial long-term survival rates support the possibility of using short-course pembrolizumab for HNSCC. Short course of pembrolizumab also provides incentives to reduce medical costs. The feasibility of short-course anti-PD-1 warrants a prospective randomized study to confirm its efficacy.


Background and Objectives: In patients with paroxysmal atrial fibrillation (AF) undergoing CABG or aortic valve surgery, many surgeons are not willing to open the left atrium to perform a complete left-sided Cox-Maze lesion set. Pulmonary vein isolation (PVI) is often favored in those patients. We investigated the outcome of patients with isolated pulmonary vein isolation compared to those receiving an extended left atrial (LA) lesion set. Materials and Methods: Between 2003 and 2016, 817 patients received concomitant surgical AF ablation in our institution. A total of 268 patients with paroxysmal AF were treated by surgical ablation concomitant to AVR or CABG. Of those, 86 patients underwent a complete left-sided lesion set, while 182 patients were treated with an isolated pulmonary vein isolation. The primary endpoint was freedom from atrial fibrillation at 12 months’ follow-up. Results: There were no statistically significant differences regarding baseline characteristics. No major ablation-related complications were observed in any of the groups. In the PVI group, three patients (1.6%) had an intraoperative stroke, while two (2.3%) patients experienced a stroke in the LA ablation group (p = 0.98). In-hospital mortality was 3.4% in the PVI group, and 2.8% in the extended LA group (p = 0.33). Freedom from AF at 12 months’ follow-up was 76% in the extended LA ablation group and 70% in the PVI group, showing no statistically significant difference (p = 0.32). Conclusion: Surgical AF ablation concomitant to CABG or AVR in patients with paroxysmal AF is safe and effective. There was no statistically significant difference between the compared lesion sets in terms of freedom from AF, survival or stroke rate after 12 months. Atrial fibrillation (AF) is the most common arrhythmia and is associated with thromboembolic events, including stroke, and is even associated with an increased mortality ,2. FurthThe initial Cox Maze procedure, which included the cut-and-sew technique, has been modified to the Cox Maze III procedure, which has been the gold standard for surgical ablation for many years . Neverthn = 26) were excluded from this analysis due to the inferiority of unipolar radiofrequency as an energy source. IRB approval was obtained (Ethikkommission der Ärztekammer Hamburg 2020-10183). An isolated pulmonary vein isolation (PVI) has been used in 182 patients, while a complete left-atrial lesion set was used in 86 patients. The choice of the lesion set was left to the discretion of the operator. The complete left-atrial lesion set included a box lesion as well as left atrial appendage- and isthmus isolation. A retrospective single center data analysis was performed. The results and outcomes of patients with PVI was compared to patients receiving a complete left atrial lesion set. Applied energy sources included argon-based cryoablatation  in 79 patients and bipolar ablation  in 189 patients.Between January 2003 and 2016, 817 patients were treated with concomitant surgical ablation in our institution. A total of 268 patients with paroxysmal AF were treated concomitant to aortic valve surgery and/or coronary artery bypass grafting and were included in this retrospective data analysis. Patients treated with unipolar radiofrequency (n = 67) or 24-h Holter ECG (n = 227) in our clinic 3 and 12 months post-surgery. Follow-up methods were equally distributed between groups (PVI group: ILR n = 48 (26.4%); extended LA group: ILR n = 19 (22.1%), p = 0.42. AF recurrence was defined by AF Burden >0.5% in ILR interrogation or duration of AF episode >30 s in 24 h- Holter- ECG. The discharge rhythm results were obtained by 12 lead ECG. The anticoagulation regimen was maintained for three months postoperative in all patients and then adapted according to CHA2DS2-VASc score. In patients without contraindications, amiodarone was used as an antiarrhythmic drug therapy, otherwise other class I or III antiarrhythmic drugs were used for at least three months postoperative and then adapted according to either ILR- or 24 h ECG rhythm results.Follow-up was conducted by either implantable loop recorder (ILR) interrogation  as appropriate. A p-value of less than 0.05 was considered statistically significant.All statistical analyses were done with SPSS statistical software version 28.0  Continuous values are displayed as mean ± standard deviation and were compared with a Students Baseline patient characteristics are shown in Performed surgical procedures included isolated coronary artery bypass grafting (CABG) in 101 patients in the PVI group and 44 patients in the extended LA group. Aortic valve replacement was performed in 48 patients in the PVI group and 24 patients in the LA group, respectively. Combined CABG and AVR surgery was conducted in 33 patients of the PVI group and 18 patients with an extended LA ablation.p < 0.001).Mean cross-clamp time was 71 ± 8 min in the PVI group and 89 ± 9 min in the extended LA group, corresponding to a significantly longer cross-clamp time in patients receiving a complete LA lesion set (p = 0.33). The one-year survival rate was 94.7% in the PVI group and 93.5% in the extended LA group, without significant differences between groups. In three (1.6%) patients in the PVI group, a perioperative stroke occurred, while two (2.3%) patients in the extended LA group experienced a stroke (p = 0.96).There were no major ablation-related complications in any of the patients. No intraoperative death was observed. In-hospital mortality was 3.4% in the PVI group and 2.8% in the extended LA ablation group (p = 0.67). During follow-up, two patients in the PVI only group experienced left-atrial flutter, while none of the patients receiving an extended left atrial lesion set had LA flutter episodes. All patients with atrial flutter were successfully treated by catheter ablation.Permanent Pacemaker implantation rates were 5.0% and 6.4% in PVI- and the extended LA group, without statistically significant differences (p = 0.22). Furthermore, at the three months’ follow-up, there was no significant difference regarding freedom from AF rate between the PVI and the left-atrial group (PVI: 70% vs. LA: 73% p = 0.54). One-year follow-up showed freedom from AF rate of 76% in patients with left-atrial ablation compared to 70% in the group of patients with PVI only, showing no statistically significant difference (p = 0.32) p = 0.49).Patients received 24 h Holter-ECG or ER interrogation at 12 months’ follow-up. There were no statistically significant differences between groups regarding freedom from AF rate at discharge . However, in this study, patients with persistent- or longstanding persistent AF were also included. In those patients, with non-paroxysmal AF, atrial fibrillation might be more often initiated from triggers outside of the pulmonary veins. Here, macro-reentrant circuits from all over the atria might contribute to initiation and the perpetuation of atrial fibrillation.In an pioneering electrophysiological study by Haissaguerre et al. in 1998, it has been shown that in stand-alone paroxysmal AF, the triggers initiating AF are mostly originating from the pulmonary veins . Since tTherefore, a more extensive ablation with a complete left-atrial lesion set or even a biatrial ablation might be necessary to achieve satisfactory results and to eliminate the non-pulmonary vein triggers and rotors. Furthermore, in patients with structural mitral valve disease, atrial fibrillation might originate from the left atrial wall itself. This might be related to the increased left atrial volume and pressure caused by the mitral regurgitation.The type of atrial fibrillation has an effect on the outcome of patients. In a recent publication by Nuzzi and colleagues in a large cohort of patients with DCM, it was shown that the type of AF influences the outcome. Patients with permanent AF had a significantly worse outcome compared to patients with paroxysmal or persistent AF .In our investigation, which was limited to patients with paroxysmal AF and without mitral valve disease, an isolated PVI achieved comparable rhythm results in the time frame of 12 months. In future, a longer follow-up might be interesting to compare the results in the long-term run and to draw definite conclusions concerning rhythm results over time. In our study, the use of ablation energies was not equally distributed between groups. In a group of patients receiving PVI, most of them were treated with bipolar radiofrequency while the majority of patients receiving the extended left-atrial lesion set were treated with cryoablation. This might have influenced the rhythm results in our study. However, in our own experience, as well as in other previously published papers, no differences in efficacy between cryoablation and bipolar radiofrequency ablation were observed.The statistically significant longer cross-clamp time, which was observed in the extended LA group, had no influence on postoperative outcome. Both lesion sets were safe and feasible, with a low number of peri-procedural complications and similar survival-rates between groups during one year follow-up. Furthermore, the rate of permanent pacemaker implantations was comparably low in both groups. However, atrial flutter was observed in two patients in the group of patients with PVI only. This might be related to the absence of a mitral isthmus line in those patients, begetting peri-mitral flutter, which is, however, a rare entity in patients with paroxysmal AF. All patients with atrial flutter were successfully treated with catheter-based ablations during 1-year follow-up.In this selected patient collective that only included patients with paroxysmal AF undergoing CABG or AVR, a pulmonary vein isolation might be sufficient to achieve satisfactory rhythm results during one-year follow-up. Furthermore, no differences between complication and survival rates were observed between groups.This study is a retrospective one with the potential risk of bias by unknown confounders. Furthermore, our series is a single-center study and is limited by the heterogeneity of our patient population, including different ablation energy sources. Additionally, the follow-up was limited to 12 months.


Background: Impaired coronary blood flow causes cardiac ischemia. Cellular therapy is a new approach to the treatment of myocardial ischemia. This study aimed to investigate the effect of adipose tissue-derived mesenchymal stem cells (AD-MSCs) conditioned with vasopressin on oxidative stress, perivascular collagen, and angiogenesis caused by myocardial infarction (MI) in rats. 6 AD-MSC received) and AVP-ASC-MI group (received 6 × 106 AD-MSC conditioned with 10 nM vasopressin). Then, histologic parameters and anti-oxidant enzymes were evaluated 7 days post-MI cell injection. We divided 40 male albino Wistar rats into 4 groups; Control group; No intervention; in experimental groups, after it generated induced MI on models, it divided into three groups: Vehicle group (150 μl of cell-free culture medium received); ASC-MI group , catalase (CAT), (GSH) Glutathione, and Total Anti-oxidant Capacity (TAC) parameters were not significantly different between the cells received groups compared with the vehicle group. But the Catalase (CAT) parameter in the ASC-MI group had a significant increase from the control group. We prepared direct evidence that intramyocardial injection of AD-MSCs reveals the positive cardiac remodeling post-MI in rats, and these useful effects can be more enhanced by administrating injection of conditioned ADSCs with vasopressin. It is the keystone of cardiac ischemia (a disorder of blood flow in the coronary arteries) . Cell thDuring ischemia, the secretion of Matrix Metalloproteinases (MMPs) disrupts, causing collagen to accumulate around the coronary arteries , 5. AD-MArginine Vasopressin (AVP) or Antidiuretic Hormone (ADH) is a potent vasoconstrictor. It has positive effects on the heart and arteries. The effect of vasopressin causes contraction and expansion of coronary arteries, and, besides, the effect on coronary blood flow has mitogenic and metabolic effects on the heart. Regulation of vasopressin secretion and action thus represents a key homeostatic process that protects the osmotic milieu of the body, allowing normal cellular function such as effect on stem cells under hypoxic conditions .  Reactive oxygen species (ROS), by regulating vascular cell function, can play a central role in normal vascular physiology and significantly contribute to the development of the cardiovascular system. ROS plays a major role in the onset and progression of cardiovascular dysfunction associated with diseases, such as hyperlipidemia, diabetes mellitus, and hypertension . We beliOxidative stress during ischemia leads to lipid peroxidation. To overcome the problem of ROS instability in measurement, malonaldehyde (MDA), a stable end product of lipid peroxidation, is often used as a marker of ROS production. Anti-oxidant scavenging systems defend organisms against ROS and inhibit oxidative damage . The catPrevious studies have found that, after MI, there is an increase in plasma MDA levels and a decrease in various anti-oxidants such as a-tocopherol, b-carotene, and vitamin C, suspend oxide dismutase and glutathione peroxidase , 13. TheAnimal model of MI6) as ASC group and animals’ recipient of the conditioned Adipose stem cells with vasopressin (6 × 106) as AVP-ASC group. All animals were given a rat chow diet, and water ad libitum and were housed under an alternating 12-hr/12-hr light/dark cycle.The experimental procedures followed the organizational guidelines for the care and use of laboratory animals and approved by Lorestan University of Medical Sciences . 40 male albino Wistar rats weighing 250 to 300 g were divided into 4 groups. Animals in the control group had no intervention (intact heart), and in experimental groups after myocardial infarction (MI), recipients of culture medium (150 ml) as vehicle group, rat recipients of Adipose stem cells  , 19. A lEquipmentEchocardiography evaluated cardiac function noninvasively ; This ultrasound system is equipped with a 7-14 MHz probe.Two-dimensional echocardiography Echocardiography was performed on animals who underwent mild anesthesia with ketamine at 10% (50 mg/kg) and Xylazine at 2% (10 mg/kg). Echocardiographic parameters were based on the main axis of the heart and were considered heart failure with an ejection fraction less than 50%. Cardiac output was estimated as (end-diastolic volume–end-systolic volume) × heart rate (ml/min)  according to the manufacturer’s protocol was injected into the ischemic area  (code number: C0889). ASCs were isolated, characterized, and maintained in culture as previously described. Under this experimental condition, 28 μl of vasopressin was dispensed in a 7 ml culture medium containing DMEM  that includes low glucose concentrations and formulations without L-glutamine, 10% FBS, 1% Pen-Strep, and diluted 100 times. In brief, Cells were recovered and plated onto 10 cm culture plates. At 24-hour intervals, cultures were washed with PBS to take contaminating other unattached cells and refed with fresh medium. This process was repeated for three days , 23. ExpSerum collection and histological sectioning2), 4. Examining fibrosis, in per arterial tissue by collagen deposition (blue). All procedures were performed in samples obtained from middle segments of the ischemic heart border zone. Results were calculated of muscle layer diameter and cardiac fibrosis using the following formula and NIH ImageJ, version 1.42q software   were collected and weighed. The serum was isolated by centrifuging at 4 °C, 15 min, 3000 rpm, and kept at -20 °C for biochemical evaluations. At the end of the procedures, hearts were rapidly removed and fixed in 10% formalin for 24 hr, embedded in paraffin, and histologically sectioned (5 mm). Samples were mounted onto slides and stained with hematoxylin, eosin, and Mason trichrome for measurement of the following histologic parameters at 400×magnification: 1. Area Occupied by Artery (AOA), 2. Muscle layer thickness in the arteries, 3. Number of capillaries in the six randomly selected fields (mmstogram) , 26.Biochemical analysisEvaluation of oxidative stress and inflammatory biomarkers et al.’s method , as the marker of lipid peroxidation, in the rat’s serum was measured based on the thiobarbituric acid (TBA) assay , 28, whis method . The meas method  with soms method  and the s method . Resultss method -38.Statistical methodspost hoc LSD test, and nonnormal distribution by the Mann–Whitney U test. Results were presented as mean ± standard error (SE) and a P-value lower than 0.05 was accepted as statistically significant. All graphs were structured using GraphPad Prism version 8.0.2. .The SPSS statistical software  was used for the analysis of data. Distributions of data in each experimental group were determined using the Kolmogorov-Smirnov test. Normal distribution data were compared using one-way ANOVA, followed by a Human AD-MSC cardiac preservation and bio-distribution  On day seven, the fluorescent-labeled AD-MSCs were found in the infarct border zone of myocardial infarction in ASC-MI and AVP-ASC-MI groups by Labomed 400×magnification EPI-Fluorescence Microscope. Evaluation of arterial density at a probe (12×12cm), using imaging of hematoxylin-eosin-stained tissue sections under a light microscope, 40×magnification was done. The Mean±SEM of Arterial density in the vehicle and control group; the lowest and the highest, respectively 0.4±0.516 and 1.1+0.56. Evaluation of Area Occupied by Artery (AOA) (mm²) revealed Mean±SEM of the vehicle and control groups; the lowest and the highest were respectively 0.02593±0.0117 and 0.05199±0.0282 . The meaBiochemical analysis P=0.6891. Mean±SEM of serum GSH concentration in the control group was 2.748±0.741, the lowest and the highest were related to the AVP-ASC-MI group at 3.687±0.739, P=0.6970. The standard deviation of the groups was between 0.23 and 0.267 and they did not differ significantly from each other. Mean ±SEM serum activities of CAT, in AVP-ASC-MI (lowest) and ASC-MI (highest) were 200.354±82.579 and 387.797±95.453, respectively, P=0.9184. The standard deviation values were between 26.1 and 39.52, which were approximately equal. The mean ±SEM of the TAC value control group was 1.4968±0.059, the lowest, and the ASC-MI group was 1.5455±0.044, the highest, P=0.6930. The standard deviation of the groups was between 0.0142 and 0.0195, showing that the groups were not significantly different. In the study of blood serum of the studied animals, the Mean ±SEM of the concentration of oxidative stress metabolite MDA was the highest vehicle average, 0.308±0.0173. The standard deviation values between the groups were 0.0025 to 0.0032, showing that the groups were not different, P=0.6891. Mean±SEM of serum GSH concentration in the control group with 2.748±0.741 was the lowest and the AVP-ASC-MI group at 3.687±0.739 was the highest, (P=0.6970). The standard deviation of the groups was between 0.23 and 0.267 and they did not differ significantly from each other. Remarkably Mean ±SEM serum activities of CAT, in AVP-ASC-MI (lowest) and ASC-MI (highest) are 200.354±82.579 and 387.797±95.453, respectively (P=0.9184). The standard deviation values are between 26.1 and 39.52, which are approximately equal. Also, the study of confidence intervals shows that the ASC-MI group has a significant difference from all groups. Also, the Mean ±SEM of the TAC value control group is 1.4968±0.059 (lowest) and that of the ASC-MI group is 1.5455±0.044 is the highest, P=0.6930. The standard deviation of the groups was numerically between 0.0142 and 0.0195 and showed that the groups were not significantly different.Concentrations of oxidative stress metabolites in blood serum were identified. The mean ±SEM of MDA concentration in the vehicle group was 0.308±0.0173, which was the highest level between groups. The standard deviation values between the groups were 0.0025 to 0.0032, showing that the groups were not significantly different, Recent studies have shown that cardiovascular risk factors, such as MI, correlate with the number and function of arterial vessels. Understanding the mechanisms that regulate endothelial cells’ function may provide new insights into the pathogenesis of vasculogenesis and may lead to the development of specific treatments to prevent ROS production and ultimately correct vascular dysfunction. We have shown that arterial histological parameters improve vascular function through anti-oxidant mechanisms. In the present study, we describe our current understanding of the contribution of oxidative stress to stem cells with vasopressin-conditioned medium and vascular dysfunction in cardiovascular disease , 40. OurEarly renovation of blood flow to the myocardium infarction is the only way to prevent progression to myocardial necrosis and thus limit the infarct size. However, a sudden renovation of oxygen supply to previously ischemic myocardium can lead to oxidative stress, with consequent oxidative injury to cells’ function and structure because of lipid peroxidation. Several studies have evaluated the level of MDA in patients with MI after treatment . In all  MDA levels in the vehicle group were higher than in the control group, and TAC levels were lower. This shows that oxidative stress is elevated during ischemia, which agrees with previous studies. We did not find any correlation between oxidative stress parameters (MDA and TAC) and serum enzyme activity, showing that these aspects of oxidative stress do not depend on the extent of damage to the myocardium .et al. did not find a direct relationship between 8-iso-PGF2a, a marker of oxidative stress, and troponin T, a marker of myocardial injury. The duration of coronary occlusion, evaluated as the time from the occurrence of the symptoms of MI to the opening of an infarct-related artery, also did not correlate with the serum MDA and CAT, GSH, and TAC levels. Similarly, Berg et al. reported similar findings. We reported an increase in MDA levels during the prolongation of ischemia in rat hearts; in our research, the duration of ischemia was evaluated by the history of pale. It is unknown whether prolonged ischemia  leads to a continuous rise in MDA levels or whether the level reaches a plateau. All previous studies reported an increase in the MDA levels following the opening of infarct-related arteries . AlthougWe found an immediate and significant reduction in TAC after MI, but in ASC-MI it was higher than in control and other experimental groups. The decrease in TAC after MI may show a depletion of anti-oxidants because of the overproduction of ROS in the damaged area.A decrease in TAC after cell therapy suggests increased ROS production, but the decrease in MDA failed to document increased lipid peroxidation in our animals. It is unlikely that additional medical treatments influenced our results. We showed the efficiency of the decrease in hydrogen peroxide levels in cases of oxidative stress in the cell cultures. Overexpression of CAT protected endothelium of the human aorta against apoptosis caused by the oxidized forms of low-density lipoproteins (ox-LDL), in our research CAT increased in the ASC-MI group, reversely in AVP-ASC-MI group it decreased. In addition, in both groups, the GSH levels were increased. In the future, these results may provide the key treatment for human heart failure; they confirm the efficacy of the combined effect of SOD and CAT activity in blocking oxidative stress. Meanwhile, the conjugation of SOD or CAT with antibody to platelet-endothelial cell adhesion molecule-1 (PECAM-1) provided a versatile molecular tool for testing the role of reactive oxygen species in vascular pathology. Anti-PECAM/SOD, but not anti-PECAM/CAT, inhibited vascular endothelial growth factor (VEGF)-induced increase in endothelial permeability. This has identified a crucial role for endogenous superoxide radicals in VEGF-mediated regulation of endothelial barrier function. Anti-PECAM/CAT, but not anti-PECAM/SOD, alleviated endothelial hyper-permeability, implicating primarily hydrogen peroxide in the disruption of the endothelial barrier. Targeting the anti-oxidant enzymes to endothelial cells offers a future perspective for the development of effective cardioprotective remedies . Our anaThe analysis of SOD activity provided further confirmation of the role of impaired anti-oxidant status in facilitating cardiovascular disease, although catalase showed no predictive value of cardiovascular events. Among the 3 anti-oxidants, GSH showed the best predictive value, as it was the only enzyme significantly associated with cardiovascular events in the fully adjusted model. For this purpose, we measured the activity of three anti-oxidant enzymes, namely glutathione (GSH) and catalase, which catabolize hydrogen peroxide 8 and superoxide dismutase (SOD), which converts superoxide anion to hydrogen peroxide 9. In our research, conditioning for transplanted cells led to increased cell survival in the tissue after transplantation. Many studies are performed on conditioning stem cells with vasopressin under hypoxic conditions. Vasopressin is a nano-peptide that is enhanced in congestive heart failure and has a protective role in heart damage. In cardiac ischemia, there is a decrease in arterial density because of hypoxia and lack of expression of angiogenesis factors. But AD-MSCs increase arterial density by having the potential to secrete angiogenesis factors and express VEGF . Hypoxiaet al. showed that the expression of the α-SMC Actin marker is present in AD-MSCs and causes the formation and increase of smooth muscle in the arteries (We showed treatment with ASCs and AVP-ASCs transplanted cells in the ischemic region would influence angiogenesis and increase the muscular diameter of the arteries. Lee arteries . Also, rThe findings of this study showed treatment with AVP-ASCs has a relatively greater effect on tissue factors in the experimental models of MI. ASCs cause growing muscle diameter and reduced collagen deposition in coronary arteries. This approach may represent a promising alternative strategy for cardiovascular disease treatment. Change in the activity of anti-oxidant enzymes in the bloodstream of experimental groups provides evidence that supports the role of the anti-oxidant system in the activation of vasopressin against oxidative stress. This result could be a defense mechanism in pathological conditions derived from myocardial infarction.FCH Supervised, conceptualized, provided methodology, analyzed or interpreted the data, and wrote, reviewed, and edited the manuscript. AN Supervised the design of the heart attack model. HA Contributed to the biochemical studies. MB Participated in the histological and biochemical studies and contributed to the statistical analysis. SN Contributed to cardiac surgery and cell differentiation technique. All authors read and approved the final manuscript.  The authors declare that they have no competing interests.


In this paper we are focusing on the action mechanism of RDV and how it can be improved in vivo. The stability of RDV alone and on encapsulation with our platform technology based polymer NV-387 (NV-CoV-2), were compared in presence of plasma in vitro and in vivo. Furthermore, a non-clinical pharmacology study of NV-CoV-2 (Polymer) and NV CoV-2 (Polymer encapsulated Remdesivir) in both NL-63 infected and uninfected rats was done. In addition, the antiviral activity of NV-CoV-2 and NV-CoV-2-R was compared with RDV in a cell culture study. The results are (i) NV-CoV-2 polymer encapsulation protects RDV from plasma-mediated catabolism in both in vitro and in vivo, studies; (ii) Body weight measurements of the normal (uninfected) rats after administration of the test materials (NV-CoV-2 and NV-CoV-2-R) showed no toxic effects. (iii) Body weight measurements and survival rates of the NL-63 infected rats were similar to the uninfected rats after treatment with NV-CoV-2 and NV-CoV-2-R. Overall, the efficacy as an antiviral regimens were found in this order as below; NV-CoV-2-R > NV-CoV-2 > RDV. Our platform technology based NV-387-encapsulated-RDV (NV-CoV-2-R) drug has a dual effect against different variants of the coronaviruses. First, NV-CoV-2 is an antiviral regimen. Secondly, RDV is protected from plasma-mediated degradation in transit. All together, NV-CoV-2-R is the safest and efficient regimen against COVID-19.Remdesivir (RDV) is the only antiviral drug approved for COVID-19 therapy by the FDA. Another drug LAGEVRIO™ (molnupiravir) though has not been approved yet by FDA but has been authorized on December 23, 2021, for emergency use to treat adults with mild-to moderate COVID-19 symptoms and for whom alternative COVID-19 treatment options are not clinically appropriate. The fact is that the efficacy of RDV is, however, limited  Nanoviricide is a platform-technology-derived biomimetic polymer that can bind to virus-specific ligands sulfanyl}-3-sulfanyl]propanoyl} oxy)-4-{[1-(hexadecylcarbamoyl)-3-[oxy]-3-oxopropyl]sulfanyl}butan-2-yl)oxy]-3-oxopropyl}sulfanyl)butanedioic acid. Based on polyethylene glycol of 22 repeat units (m) substituted by two hexadecylamine (R1), the theoretical molecular formula for the repeat unit of the polymeric drug substance  is C104H188N2O44S4. Nominal calculated formula weight of the polymer repeat unit (RU) is 2298.85 g/mol when the hexadecylamine substitution level (x) per repeat unit is 2, the MMSA substitution level (y) is also 2, and the starting polyethylene glycol  consists of 22 repeat units (m). The actual substitution levels are less than these theoretical limits and are described in the drug substance specifications. The repeat unit (RU) formula weight at hexadecylamine (HDA) substitution level x between 0 and 2 is described by the formula: MWRU = 1853 + 223x. The degree of polymerization, n, in P10M2DT (HDA)x (MMSA)y polymer is 8 ± 2 . The rats were up to 6 weeks old and ranged between162-199 in their body weight. Each group contains 6 animals ; NV-CoV-2 Medium (50 mg/kg); NV-CoV-2 high (100 mg/kg). The drug (5ml/kg) was injected through IV.There was an evaluation of pulmonary and neurobehavioral functions following intravenous administration of NV-CoV-2 in 24 conscious male rats  . The baseline respiratory parameters were obtained for 5 minutes, followed by a stabilization period. Each rat was removed from the chamber and dosed based on their assigned test groups. After the treatment dose was administered, the rat was returned to its designated plethysmograph chamber and allowed to stabilize. The respiratory parameters were recorded again but measured for 5-minute intervals at 15 min, 1 hour, 2 hours and 4 hours time points. Respiratory parameters were measured at ±5 minutes for the 1 hour evaluation and ±15 minutes for the 1, 2 and 4 hours post-dose evaluations. After each reading, the animals were removed from the plethysmograph chamber and returned to the home cage. The following parameters were recorded (SOP PHA-SOF-90) using the Ponemah Physiology Platform (Ponemah v.5.20 Pulmonary): Respiratory rate; Tidal Volume, and Minute Volume .An modified Irwin and functional observations battery tests were used for the neurobehavioral evaluation. At approximately 15 minutes, 1 hour and 4 hours after treatment dosing and the completion of the pulmonary evaluation was finished, the animals were placed in a fixed environment. The rats were separated into groups and placed in a Plexiglas® enclosure with a fitted lid. The enclosure was placed on absorbent paper that detects excretions. In this environment, the rats were free to move about and interact with the other rats.The observations were recorded for the presence of the following symptoms of the group in the Plexiglas® enclosure. The animal number was documented when any of the following symptoms were observed. These symptoms included seizures/convulsions (awareness reaction), body tremors (motor activity), ataxia (piloerection) and abnormal posture (stereotypy). Excretion (decreased respiration) was documented separately as a group observation , 33.The different tests done on all the animals were as followed. Clapping the hands immediately above the open square and watching the response and reaction tested for the startle response. Each animal was individually removed from the square and examined for vocalization, irritability and increased secretion of saliva or tears. Changes in abdominal tone were also recorded by feeling the rat’s abdominal muscles. The loss of the righting reflex was examined by placing each animal on its back and monitored how they behaved. Pupil size was recorded for mydriasis or miosis under a magnifying glass. An increase or decrease in their nociceptive response was tested by a tail pinch. A cotton ball and blunt probe checked the corneal reflex and pinnal reflex. Each animal was tested for immobility and grip strength on an inclined wire screen.In addition, rectal body temperatures were taken from all the animals approximately 1 and 4 hours after dosing using a YSI Precision Digital Thermometer (Model 4600).Macaca fascicularis); Total Number /Sex): 4 Male; Age Range: 2–5 years; Body Weight Range: 5.1 to 6.0 kg prior to treatment). The test article administration and group assignments are shown below in There was an evaluation of the cardiovascular function after administration of NV-CoV-2 in conscious telemetered cynomolgus monkeys . There were 4 male monkeys, between the ages of 2 to 5 years, which weighed approximately 5.1 to 6.0 kg prior to treatment  minutes by a percutaneous IV catheter. Animals were restrained during dosing to prevent any movement. Prior to dosing, all the animals were trained on the restrainers.Data was collected for 24 hours before the first dosing day in order to establish acceptable baseline parameters for each animal. The baseline data was retained in the study file, but not in the final report. Pre-dose values were determined from data that was collected at least 24 hours prior to each subsequent dose given to the animals. Following the dose administration, parameters were recorded continuously for 24 hours. A washout period of approximately 6 days was allowed between test article doses .The following parameters were analyzed using the Ponemah 5.20 SP9 and ECG Analysis Module v. 5.30 software (SOP PHA SOF-106): Arterial Blood Pressure, Heart Rate (HR), ECG, and Body Temperature . One-minSince NV-CoV-2 was administered through an IV injection, it is important to determine if there is any evidence of immunogenicity. A study was conducted for detecting any anti-NV-CoV-2 antibodies in the rat serum samples. These serum samples were collected on day 14 and 28. NV-CoV-2 was given to the animals in 6 sequential IV injections over a 10-day period. In order to quantify and detect any anti-NV-CoV-2 antibodies in the serum samples, an Indirect ELISA assay was preformed , 37. Thi2 asphyxiation. The primary end points were survival and loss of body weight.The entire animal handling experiments were done by Dr. Krishna Menon from AR Biosystems, , based on the protocol #IACUC No. 14/17ARB. This study followed strict guidelines and recommendations from the “Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.” All surgeries were performed under sodium pentobarbital anesthesia in order to minimize suffering. There aren’t any known acceptable alternatives to the use of live animals to accomplish the purpose of this study. The large historical database concludes that animal study is a standard model for non-clinical studies. In fact, previous investigations with the same disease models found this to be the most suitable model. The rats were observed daily for body weight, clinical and behavior changes. At the end of the experiment, the rats were sacrificed by COThis study was designed to use the fewest amount of animals possible. The amount of animals used was consistent with the scientific objectives of the study, the contemporary scientific standards and for applicable regulatory requirements. The number of animals used is considered the minimum necessary for meaningful biological comparisons and statistical calculations.The t-test and one-way analysis of variance were used for statistically analyzing the data. Percentages of variation for all values are below 10%.The analytical method used for detection and quantification of NV-CoV-2 in plasma samples was direct ELISA. The result shows that for all doses, NV-CoV-2 was detected in rat plasma producing an initial increase that peaked between 4–8 hours and decrease below the detection level between 24 and 48 hours .≤ NV-CoV-2-R < NV-CoV-2. For the cells, LLC-MK2, the order of antiviral capacity are: RDV = NV-CoV-2-R = NV-CoV-2. PBS and DMSO, as a negative control do not show any antiviral capacity in either case.Dose-responsive antiviral effects of NV-CoV-2, NV-CoV-2-R and RDV were observed as a protection of MRC-5 cells or LLC-MK2 cells infected with CoV-229E virus (A) or CoV-NL63 (B) virus, respectively. Antiviral efficacy is in the following order with MRC-5 cells: RDV The untreated and vehicle-treated rats infected with the CoV-NL63 virus survived up to day 5. Rats treated daily with RDV (10 mg/kg) survived 7.5 days. The rats treated with NV-CoV-2 (160 and 320 mg/kg) survived until 13.5 and 14 days. Therefore, treatment with NV-CoV-2 extended the survival of rats infected intra-tracheally from the lethal dose of the human CoV-NL63 virus. More importantly, NV-CoV-2 treatment was better than RDV treatment alone.The survival of rats with the lethal dose of CoV-NL63 was dependent on the number of days of the drug used for the treatment. An increase in the number of days of treatment correlated with an increase in the survival days. The survival of the rats administered with NV-CoV-2 for 1 day was similar to the untreated rats and vehicle-treated rats of 6–7 days. When the treatment injection was for 3 to 5 days, the survival increased to 13.4 to 17.6 days.In vitro, NV-CoV-2 polymer encapsulation protects RDV from plasma-mediated catabolism  injections, both 1st and 5th. In brief, NV-CoV-2 polymer can encapsulate Remdesivir efficiently.NV-CoV-2 is capable of encapsulating other drugs , 10. Thitabolism . In vivoSurvival rate of NL-63 infected male rats after treatment with NV-CoV-2 encapsulated RDV (NV-CoV-2-R). The group of rats treated with NV-CoV-2-R had a slight increase in survival after 1 day of treatment of 8.8 days. The vehicle-treated rats only survived 7.7 days. When NV-CoV-2-R injection was for 3 to 5 days, the survival increased to 13.9 and 18 days  and high dosed (320 mg/kg/injection) NV-CoV-2 treated rats significantly lost 25.7 and 12.6 g in body weight.As a result, the treatment with NV-CoV-2 for both 160 and 320 mg/kg/injection  on days 0, 1, 3, 5 and 7 had significant effect on the survival in the rats infected with the human coronavirus CoV-NL63. Therefore, NV-CoV-2 treatment against the human coronavirus infection is clearly the superior treatment than RDV.Effects of the vehicle and NV-CoV-2  on pulmonary parameters are shown in the The intravenous administration of NV-CoV-2 at 25, 50 or 100 mg/kg did not induce any significant effect on respiratory rate, tidal volume or minute volume when compared to the vehicle control group.As shown in the Body temperature was not affected by the intravenous administration of NV-CoV-2 at 25 or 50 mg/kg at 1 or 4 hours post-dose when compared to the vehicle control group . Body teRandom occurrence of fluctuations in blood pressure and heart rate are inherent in conscious freely moving animals. Hence consistent (2 or more time points) changes of 15% or more from vehicle controls were considered a significant  change for blood pressure and heart rate. No significant effects on blood pressure  or heart rate were observed after the intravenous administration of 25, 37.5 or 50 mg/kg doses of NV-CoV-2 (data not shown).All monkeys maintained sinus rhythm throughout the study. The intravenous administration of 25, 37.5 and 50 mg/kg NV-CoV-2 did not induce any significant effects on quantitative ECG parameters, as measured using the analysis software, in conscious telemetered cynomolgus monkeys.This assay detects all relevant Immunoglobulin (Ig) isotypes. Since a non-mucosal route of administration was employed and there was no indication of anaphylaxis, the relevant isotypes would be IgG and IgM. The above screening assay is validated to determine lower limit of detection level of isotopes IgG and IgM as a group, and is scientifically justified for quantification of IgG and IgM as a group, using a linear calibration curve. This ELISA has a reliable lower limit of detection of 15ng/ml at which all relevant Ig Isotope’s are clearly distinguishable from negative control, and quantifiable with acceptable accuracy and precision.Immunogenicity determination was done on all animals (4/sex/group) in duplicate following I.V. dosing of NV-CoV-2 in rats with serum samples collected at days 14 and 28 days following the start of the 6 sequential doses over 10 day period. The mean OD of all dose groups is not statistically distinguishable from the vehicle group, both sexes in both Day 14 and Day 28 animals. Under the conditions of the assay method, IgG and IgM antibodies against NV-CoV-2 were not detected and, therefore NV-CoV-2 was not shown to be immunogenic.The intravenous administration of NV-CoV-2 at doses of 25, 50 and 100 mg/kg did not affect respiratory function in rats. No significant neurobehavioral effects were observed in all dose groups of rats at the 1-hour post-dose observation. Decreased activity  and decreased abdominal tone  were observed in a small number of animals treated with NV-CoV-2 at 4-hour post-dose observation that was not dose-dependent. Body temperature in rats was not affected by the intravenous administration of NV-CoV-2 at 25, 50 and 100 mg/kg.The intravenous administration of NV-CoV-2 at doses of 25, 37.5 and 50 mg/kg in conscious telemetered cynomolgus monkeys did not induce any significant, biologically relevant effects on heart rate, arterial blood pressure, cardiac rhythm, and ECG parameters. All monkeys maintained sinus rhythm throughout the study.Paramyxoviridae, Pneumoviridae and Coronaviridae families with the EC50 in the sub-micromolar range Reviewers' comments:Reviewer's Responses to Questions  Comments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?  The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********  2. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #1: YesReviewer #2: Yes**********  3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #1: YesReviewer #2: Yes**********  4. Is the manuscript presented in an intelligible fashion and written in standard English?  PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********  5. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Results needed to be explained better. They have made statement on the basis of unpublished a number of times. Why they have not shown data. Discussion did not discuss results in very effective manner. Discussion does not detailed similar studies. They have not compared their results with other studies.Reviewer #2: The manuscript is well-written and well-presented. Besides, the study is well-designed. Only comment is that the authors claimed that “Remdesivir (RDV) is the only antiviral drug approved for COVID-19 therapy by the FDA”, indeed molnupiravir is also approved by FDA as anti-COVID-19 drug.**********  6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.  Yes: Muhammad Nouman MughalReviewer #1: Reviewer #2: No**********https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  19 Oct 2022Rebuttal Letter: Oct 19, 2022PLOS ONE Decision: Revision required [PONE-D-22-24874] -Journal requirements:When submitting your revision, we need you to address these additional requirements.1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found athttps://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf andhttps://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdfAu: Done accordingly2. We note that you have stated that you will provide repository information for your data at acceptance. 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The conclusions must be drawn appropriately based on the data presented.Reviewer #1: YesReviewer #2: Yes________________________________________2. Has the statistical analysis been performed appropriately and rigorously?Reviewer #1: YesReviewer #2: Yes________________________________________3. Have the authors made all data underlying the findings in their manuscript fully available?The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.Reviewer #1: YesReviewer #2: Yes________________________________________4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes________________________________________5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Q#1: Results needed to be explained better. They have made statement on the basis of unpublished a number of times. Why they have not shown data. Au: Those ‘data not shown’ part are the preliminary observation to standardize the materials and methods, and has little or no connection with the main paper, hence deleted from this revised version, also, the number of Figs remains within the limit.Q#2: Discussion did not discuss results in very effective manner. Discussion does not detailed similar studies. They have not compared their results with other studies.Au: Discussion part is now re-written accordingly, and English corrections were also done.Reviewer #2: The manuscript is well-written and well-presented. Besides, the study is well-designed. Only comment is that the authors claimed that “Remdesivir (RDV) is the only antiviral drug approved for COVID-19 therapy by the FDA”, indeed molnupiravir is also approved by FDA as anti-COVID-19 drug.Au: Thanks for updating with the new Information, and accordingly we have edited our Text ; and also in the Discussion Section, Lines showed with Blue highlightsHowever, to exchange the information:https://www.lagevrio.com/patients/?utm]LAGEVRIO™ (molnupiravir) has not been approved, but has been authorized on December 23, 2021, for emergency use by FDA, for the treatment of mild-to-moderate COVID-19 in adults who are at high risk for progression to severe COVID-19, including hospitalization or death, and for whom alternative COVID-19 treatment options authorized by FDA are not accessible or clinically appropriate [6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). 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The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********  3. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #1: YesReviewer #2: Yes**********  4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #1: YesReviewer #2: Yes**********  5. Is the manuscript presented in an intelligible fashion and written in standard English?  PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********  6. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: (No Response)Reviewer #2: (No Response)**********  7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). 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Mitral valve repair (MVP) is the gold standard treatment for degenerative mitral regurgitation. With the expansion of transcatheter technologies, this study compares the outcome of MVP in low-risk and non-low-risk patients to serve as a benchmark.This retrospective, single-institution study examined all patients who underwent MVP for primary mitral regurgitation from 2005 to 2018. Patients were stratified into 2 risk categories: low-risk [Society of Thoracic Surgeons (STS) Predicted Risk of Mortality (STS-PROM) ≤2%] and non-low risk (STS-PROM > 2% or age > 75), with a subgroup of very low risk .P < 0.01). For the low-risk group, the observed-to-expected (O:E) STS mortality ratio was 0.4 and the composite morbidity and mortality ratio was 0.6, whereas for the non-low risk, the O:E mortality was 1.5 and the composite morbidity and mortality was 0.9. When the subgroup of very low-risk group was assessed, the mortality O:E ratio was 0.A total of 1207 patients were included, and 1053 patients were classified as low risk and 154 as non-low risk. The non-low-risk group was significantly older, more likely to be female, and had a higher comorbidity burden than the low-risk group  is the most common valvular disease in the USA, with the burden expected to double by 2030 due to the ageing population [1]. Mitral regurgitation (MR) is the most common valvular disease in the USA, with the burden expected to double by 2030 due to the ageing population . For degWe have previously seen the expansion of transcatheter technologies from prohibitive surgical risk patients to low-risk patients with transcatheter aortic valve replacements , 10. TEEThis study was approved by the Mass General Brigham Institutional Review Board with waived informed consent .From January 2005 and December 2018, all patients with a diagnosis of primary MR who underwent isolated MVP, or MVP with concomitant tricuspid valve repair or Maze procedure, at a single institution were included in the study. Patients with a diagnosis of rheumatic mitral valve disease, secondary MR, active infective endocarditis, prior cardiac surgery and those undergoing other concomitant cardiac procedures were excluded. Patients were then stratified into 2 risk categories based on age and STS Predicted Risk of Mortality (PROM) scores. For each patient, the STS-PROM was calculated based on the published STS risk model for isolated MVP , 14 at tPrimary outcomes included the observed values and observed-to-expected (O:E) ratios for operative mortality and composite morbidity and mortality. Composite morbidity and mortality was defined as the occurrence of any one of the following: operative mortality, stroke, renal failure, prolonged intubation, mediastinitis/deep sternal wound infection or reoperation. Secondary outcomes included stroke, renal failure, prolonged intubation, mediastinitis/deep sternal wound infection, reoperation and length of stay. The definitions for these outcomes were identical to the STS definitions used in their risk prediction models and are found in The observed values for these outcomes were obtained on chart review. The expected values for each of the STS outcomes were calculated for each individual patient with their preoperative comorbidity burdens using the STS model that was applicable for the year the patient was operated on. Over the course of the study, STS versions 2.5, 2.6, 2.73, 2.81 and 2.9 were used. O:E ratios were calculated by dividing the incidence of the observed outcome by the mean expected incidence for each outcome.T-tests for normally distributed variables and Wilcoxon rank sum or Kruskal–Wallis tests if non-normally distributed. These are presented as mean and standard deviation or median and interquartile range as appropriate. Categorical variables are presented as number and percentage and were compared using Fisher’s exact test. Analyses compared the observed outcomes, expected outcomes, as well as O:E ratios for each of the 7 STS outcomes for the low- and non-low-risk groups. Kaplan–Meier cumulative survival analysis was also performed as a supplemental analysis and is provided within the Continuous variables were tested for distribution and compared using To examine the trends of observed and expected outcomes over time, the observed rate and the mean STS risk score, along with 95% confidence intervals for operative mortality and composite morbidity and mortality, were calculated for each quarter (3 months) during the study period resulting in data points for a total of 56 quarters. The upper and lower confidence limits for expected rates and the mean rate of observed outcomes were plotted, and interpolation lines calculated. This allowed for the comparison of our observed rate to expected rates while presenting any changes over time in patient complexity and expected outcomes. The trend in the mean rate of observed outcomes was then compared to the overall rate for the specific outcome, to evaluate the absolute change in postoperative morbidity and mortality over time.P-value of ≤0.05 was the criterion for significance for all statistical tests. Data were analyzed from October 2020 to April 2021.All analyses were conducted using SPSS version 26.0  or R version 3.4.1 . A two-sided A total of 1207 patients were included in the study. Of those patients, the mean age was 59.9 years, 37.7% were female and 93.5% had moderate or greater MR. The overall STS-PROM was 0.84% and the predicted STS composite morbidity and mortality was 9.43%.P < 0.001) compared to the low-risk patients. Full patient characteristics and predicted outcomes are presented in Table When stratified by surgical risk, 1053 patients were characterized as low risk and 154 patients were characterized as non-low risk. The non-low-risk patients were significantly older (78.5 vs 57.2 years) and more likely to be female (50.0% vs 35.9%) and had a significantly higher mean STS-PROM (2.93% vs 0.54%) , with stroke being the second most common (1.7%).P < 0.05) compared to the non-low-risk group. There was no significant difference in reoperation or 30-day readmission. Full intraoperative characteristics and postoperative outcomes are presented in Examining by patient risk group, the non-low-risk patients had a significantly higher proportion of urgent and emergent procedures. The low-risk group had significantly lower incidences of mortality 0.2% vs 4.5%), stroke (1.2% vs 4.5%), prolonged ventilation (2.9% vs 8.4%), renal failure (0.6% vs 2.6%) and composite morbidity and mortality , Kaplan–Meier survival curves are presented in There was no significant difference in cumulative survival between the low- and non-low-risk groups  for mortality (0.4), deep sternal wound infection (0), reoperation 0.3), prolonged ventilation (0.8), renal failure (0.5) and composite morbidity and mortality (0.6). Observed major stroke  was lower than expected with O:E ratio 0.9, although overall stroke rate was higher than expected with O:E 1.9. For the non-low-risk patients, observed outcomes were better than expected for reoperation (0.2), prolonged ventilation (0.8), renal failure (0.5) and composite morbidity and mortality (0.9). However, the observed incidence of mortality (O:E ratio 1.5), deep sternal wound infection (8.8), overall stroke (2.4) and major stroke (1.7) were worse than expected. The full observed and expected outcomes, as well as O:E ratios, are presented in Table , prolongA subgroup analysis was conducted examining a very low-risk subgroup (STS-PROM <1% and age ≤75 years) that consisted of 936 patients compared to low-risk and non-low-risk patients. The very low-risk group had a mortality O:E ratio of 0 compared to the new low-risk group of 1.31. The low O:E values of the low-risk group were mainly driven by the superior outcomes in the very low-risk group—full results of the very low-risk subgroup analysis are presented in Table In this longitudinal study that examined the observed and expected morbidity and mortality following MVP in low-risk patients, we report several significant findings. First, for low-risk patients, the observed outcomes following MVP are better than expected for mortality and composite morbidity and mortality. Second, the observed rate of composite morbidity and mortality has trended down over time, staying below the 95% confidence interval for expected values in the latter half of the study Fig. . FinallyThe low-risk patients in our study had excellent outcomes with a mortality rate of 0.2% and composite morbidity and mortality rate of 5.1%. MVP has had a decreasing mortality rate over the last several decades, with recent studies demonstrating operative mortality rates of 1.1–3.5% , 15, 16.Due to our study population’s bias towards the low-risk group, we identified a very low-risk subgroup (STS-PROM <1% and age ≤75 years) that had 0% mortality and 4.8% composite morbidity and mortality, with O:E ratios 0 and 0.65, respectively. This demonstrates that the excellent outcomes seen in the low-risk group were mainly driven by the very low-risk subgroup. In our study, the original low-risk group had similar O:E patterns as the non-low-risk group , which questions the use of STS-PROM of 2% as the cutoff for intermediate-risk population. With higher comorbidity burdens, longer lengths of stay and worse than predicted outcomes, we believe that there will be a role for transcatheter interventions within this non-low-risk group. TEER has progressively demonstrated acceptable outcomes for decreasing risk groups—from the EVEREST II trial (minimum STS-PROM = 12%) , the COAIt is important to discuss stroke, which was the only postoperative morbidity that demonstrated worse than expected outcomes for all risk groups. The overall observed stroke rate of 1.7% in our study is similar to what has been previously reported , 19. OurThe landscape of cardiac surgery has shifted in the last several decades with the advent of transcatheter technologies for coronary revascularization, aortic replacements and valve replacements/repairs. The careful evaluation of the risks and benefits of these new technologies compared to their surgical gold standards is of the utmost importance. As TEER continues to explore studies looking at lower-risk patients, the observed outcomes following MVP for each risk group should be the benchmark for acceptable postoperative outcomes. Our study’s excellent outcomes of MVP in low-risk, especially the very low-risk patients, indicate that future transcatheter technology must be trialed cautiously and benchmarked against contemporary national outcomes for these groups before further dissemination.There are several limitations in this study that need to be addressed. This study is a single institution, retrospective, observational study and has the associated limitations of such that include but are not limited to small sample size, selection bias, limited generalizability and unmeasured confounding. In addition, this study is based out of a quaternary, academic care centre that is a comprehensive valve centre, which limits the generalizability of these results to experienced institutions and may not reflect national outcomes. Second, although STS publishes modifiers for PROM adjustment, the scores used in this analysis did not use these modifiers. However, 5 different versions of STS  were used for expected risk calculation to account for the improvement in MVP outcomes over time. Finally, our study population was limited to patients undergoing MVP and not limited to patients who were intended for MVP and ultimately underwent replacement, which could introduce a selection bias. Nonetheless, this is one of the first studies to examine the observed vs expected morbidity and mortality following MVP and provides valuable contemporary outcomes for future transcatheter devices.The observed mortality and composite morbidity and mortality following MVP were better than expected for low-risk patients, particularly for the very low-risk groups that demonstrated excellent outcomes. With the future expansion of transcatheter technologies into the intermediate- and potentially low-risk groups, future studies on the national, contemporary outcomes of MVP should be the benchmark for ongoing and upcoming transcatheter device trials. While there appears to be a role for transcatheter devices in improving postoperative outcomes for non-low-risk patients, careful evaluation and the cautious study of this technology in lower-risk groups will be exceedingly critical for patient care.ICVTS online.This research did not receive any additional funding.Conflict of interest: Tsuyoshi Kaneko is a consultant for Edwards Life Sciences, Medtronic, Cook Medical, 4C Medical and CardioMech and a speaker for Abbott and Baylis. Sameer Hirji is a consultant for Cardiac ERAS Interactive Audit System (EIAS) for ENCARE. The other authors report no conflicts of interest.ivac241_Supplementary_DataClick here for additional data file.


Better NabIR were found in wild-type SARS-CoV-2 (WT) compared to Delta-type (DT) and Omicron-type (OT). Women with two-dose vaccinations demonstrated greater NabIR than those with a single dose. The cut-off value of the SRBD IgG level by the LFI-SR for NabIR to DT  was 60.15 and 150.21 ng/mL for mothers (both p = 0.005), and 156.31 (p = 0.011) and 230.20 ng/mL (p = 0.006) for babies, respectively. An additional vaccine booster may be considered for those mothers with SRBD IgG levels < 60.15 ng/mL, and close protection should be given for those neonates with SRBD IgG levels < 150.21 ng/mL, since there is no available vaccine for them.The focus of this study was to investigate the detection of neutralizing antibodies (Nabs) in maternal serum and cord blood as the targeted samples by employing a lateral flow immunoassay combined with a spectrum reader (LFI-SR) and the correlation of Nab protection against different types of SARS-CoV-2. We enrolled 20 pregnant women who were vaccinated with the Moderna (mRNA-1273) vaccine during pregnancy and collected 40 samples during delivery. We used an LFI-SR for the level of spike protein receptor binding domain antibody (SRBD IgG) as Nabs and examined the correlation of the SRBD IgG concentration and Nab inhibition rates (NabIR) via enzyme-linked immunosorbent assays (ELISA). The LFI-SR had high confidence for the SRBD IgG level ( Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,4. The vPregnant women may be at higher risk of developing severe illness while infected with COVID-19 ,8. A sysIt is important to provide rapid detection of the Nab level or SRBD IgG concentration in order to determine whether vaccine protection is still sufficient. Lateral flow immunoassays have been developed after the evolution of radio-immunoassays, enzyme immunoassays, and lateral flow technology since the 1960s . Using lThe aim of this current study was to examine women and their delivered neonates, and compare the serum SRBD IgG level results provided via the use of a lateral flow immunoassay in combination with a spectrum reader to the results provided via a traditional Nab inhibition rate evaluation using enzyme-linked immunosorbent assay (ELISA). The focus of this effort was to then determine the relative performance of a rapid test strip technique for detecting the effectiveness of vaccines against different types of SARS-CoV-2 among these vulnerable cohorts.This prospective study was performed at Kaohsiung Medical University Hospital. All participants were enrolled during admission before delivery, and all were confirmed SARS-CoV-2 infection-negative via a nasopharyngeal swab polymerase chain reaction test at the time. Patients with preterm labor were not enrolled in our study. Furthermore, all enrolled participants had singleton pregnancies, 17 of them received the second dose of the Moderna (mRNA-1273) vaccine between the 27th and 38th weeks of gestation, and all were confirmed to not have any COVID-19-related symptoms during pregnancy. Vaccinations were performed during pregnancy, and all participants were ≥20 years of age with no medical history of disease requiring immunosuppressant treatment.We collected peripheral blood and umbilical cord blood as maternal and fetal blood samples, respectively. Both blood samples were obtained on the day of birth, and the fetal blood samples were collected from the umbilical cord after cord clamping. Furthermore, all associated clinical data, including enrolled age, body mass index (BMI), weeks of vaccination, delivery weeks, and other available information, were obtained from the electronic medical record system. This prospective study was conducted with the approval of an institutional review board (IRB) (IRB number: KMUHIRB-SV(II)-20210087).All patients in this study had singleton pregnancies without symptoms related to COVID-19, and all were voluntarily vaccinated against COVID-19 during pregnancy. The exclusion criteria were as follows: (1) age < 20 years; (2) COVID-19 vaccination before pregnancy; (3) preterm labor; and (4) disease with immunosuppressant treatment.We used a SARS-CoV-2 rapid test strip to detect spike protein receptor binding domain (SRBD) IgG in maternal serum and neonatal cord blood. Here, we tested the SRBD complex instead of S1RBD IgG, since the S1RBD IgG amount in the sample may not be enough to demonstrate significantly visible results. Additionally, S1RBD IgG was used in the Enzyme-linked Immunosorbent Assay (ELISA) mentioned in a later section for precise assessments. The commercialized rapid test strip  employs a lateral flow immunochromatographic methodology with a strip containing spike protein receptor binding domain (SRBD) antigen-coated gold particles. The size of the colloidal gold used to make our lateral flow immunoassay was about 40 nm, and is commercially available from BBI Solution (Cat. No.: EM.GC40). The targeted neutralizing antibodies (Nabs) in serum were detected by a double-antigen sandwich lateral flow immunoassay. As demonstrated in The serum sample of approximately 25 µL and one drop of buffer (about 40 µL) was dropped into the sample well sequentially, where it was allowed to migrate along the strip via capillary movement. Recombinant viral SARS-CoV-2 protein (SRBD) conjugated with gold colloids was precoated on the conjugated pad to provide a colorimetric presentation. Recombinant SARS-CoV-2 protein (SRBD) was precoated at the test line. If there were Nabs in the serum sample, it conjugated with the SRBD–gold particles that were then captured and formed a complex with the SRBD at the test line, where a visible red line was generated. If, however, there were no Nabs in the serum sample, the SRBD–gold particles were not carried by Nabs, and no red line appeared at the test line region. We also placed the rapid test strip results into the spectrum analyzer to demonstrate the results shown in As shown in As shown in Since the spectrum at 650 nm was not absorbed by colloidal gold, the reflectance value of the red line for 650 nm was about 1. From this formula, if the sample had a higher SRBD IgG concentration, there would be a more obvious red color line in the test line region, and the reflectance value of the red line with a spectrum at 430–600 nm would be lower, and we would obtain a higher α value. Therefore, by the same principle, if there is a lower SRBD IgG concentration in the sample, the reflectance value at 430–600 nm will be higher, and the detected α value will be lower. In other words, the higher the α value, the stronger color intensity of the test line in the strip that indicated higher SRBD IgG concentration.We used sandwich ELISA to detect S1RBD IgG antibody to validate the rapid test strip performance. The ELISA-based detection of S1RBD IgG antibody was performed using a commercialized human IgG ELISA kit  under the manufacturer’s instructions. The samples and positive control were added to a 96-well plate coated with S1RBD protein after dilution. After 1 h of incubation at room temperature, the wells were washed and biotinylated anti-human IgG antibody was added. After 30 min of incubation, the wells were washed again and HRP-conjugated streptavidin was added. The wells were washed again after another 30 min of incubation, and TMB substrate solution, as well as stop solution, were subsequently added in the dark to see whether or not there was a colorimetric change from blue to yellow. Color intensity was measured at 450 nm via an OD reader .Linear regression was also used to determine the performance of the lateral flow immunoassay test strip/spectrum analyzer using a standard concentration gradient to determine the rapid test strip SRBD IgG value. The linear regression was used to validate the performance of the test strip and obtain a trend line equation. Furthermore, the limit of detection (LOD) and the limit of quantification (LOQ) were calculated using the standard deviation. The LOD and LOQ were considered references of sensitivity and efficacy for the lateral flow immunoassay test strip/spectrum analyzer. These calculations were performed using the following formula:The ELISA-based neutralizing antibody inhibition test can be used to detect neutralizing antibodies specific to different types of SARS-CoV-2 in serum. Here, we used a commercially available test kit for wild-type , Delta-type , and Omicron-type  viruses and performed the protocol based on the manufacturer’s instructions. All of the above kits had high precision as Intra batch CV% (coefficient of variation) < 15% and Inter batch CV% < 15% with nearly 100% high specificity. The 96-well plates were precoated with recombinant protein ACE2 for wild-type SARS-CoV-2 Nab detection and spike proteins  for Delta-type and Omicron-type SARS-CoV-2 Nab detection, respectively. The samples and controls were diluted with wash buffer and added to the wells of the 96-well plates, followed by horseradish peroxidase (HRP)-conjugated receptor binding domain solution, and a 30-min incubation time at room temperature.For wild-type Nab detection, only 15 min of incubation at room temperature was required, and supernatant removal with well washing was performed as per the provided protocol. For Delta-type and Omicron-type Nab detection, an hour of incubation at room temperature was required, and wells were subsequently washed five times, followed by the addition of ACE2-HRP solution, an additional hour of incubation, and a repeat of the washing steps. Finally, tetramethyl benzidine (TMB) substrate solution was added to all wells in the dark and the plates were incubated for 15 min before stop solution was added. The subsequent colorimetric change in each well was detected based on the O.D. value absorbance at 450 nm as read by a microtiter plate reader .The Nab inhibition percentage was then calculated with the O.D value results based on the following formula:p < 0.05 was considered to be statistically significant.To verify the rapid test strip performance, the Spearman rank correlation coefficient was determined to analyze the correlation between the results from conventional ELISA and the results from the lateral flow immunoassay/spectrum analyzer for the maternal and fetal cord blood samples. An analysis was also performed to examine the differences in Nab values for wild-type or Delta-types of SARS-CoV-2 as determined by ELISA and by lateral flow immunoassay. The data were analyzed using GraphPad Prism, and the result of A total of 20 pregnant women and 20 corresponding, pending newborns were enrolled in our study. All enrolled pregnant women were confirmed negative for COVID-19 infection during admission for delivery. The participants’ characteristics are provided in p = 0.062; neonates: 97.11% vs. 43.33%, p = 0.024). The Nab inhibition rates for Delta-type SARS-CoV-2 were quite low in maternal serum and neonatal cord blood for those with only one dose of the vaccine , and less than the cut-off value of 30% for the presence of Nabs [p < 0.001; neonates: 64.21% vs. 1.44%, p < 0.001). For wild-type SARS-CoV-2, no obvious difference in Nab inhibition rates for maternal serum or neonatal cord blood was detected during the interval between the second vaccine dose and delivery , but for Delta-type, there was a trend toward higher Nab inhibition rates when the interval was longer . Generally, Nab inhibition rates were significantly lower for Delta-type SARS-CoV-2 than wild-type in both maternal serum and neonatal cord blood . The results are shown in  of Nabs . Howeverp = 0.010; neonates: 9.28 ng/mL, p = 0.007). Similar results were found via ELISA for both mothers , and neonates . The correlation between the two detection methods was highly relevant with significance in both maternal serum  and neonatal cord blood . All results are provided in p value < 0.001), respectively, which both showed positive relevancy.p < 0.001) and 97.06% (p < 0.001), respectively. Samples from the three subjects that had only received one dose of the vaccine demonstrated significantly poorer Nab inhibition rates than the subjects that received two full doses of the vaccine . Among the subjects that received two vaccine doses, 88.2% and 82.3% of mothers and neonates, respectively, had Nab inhibition rates over 30%. As shown in p = 0.005) (p = 0.011) (p = 0.005) (p = 0.006) (p < 0.0001). p value no significance), respectively.= 0.005) A and 156= 0.011) B for mot= 0.005) C and 230= 0.006) D for motp < 0.001).The COVID-19 pandemic has produced a massive influence and impact on the economic and daily life of people, societies, and countries worldwide. As of January 2022, over 300 million people have become infected and five million people have succumbed to the disease . The devThe examined cohorts were different between the present study and our previous study . In the p < 0.0001; neonates: Rho = 0.8135, p < 0.0001). Beharier et al., reported that SRBD IgG transfer across the placental barrier can be triggered by the BNT162b2 mRNA vaccine within 15 days after administration of the first vaccine dose [p < 0.0001) [p < 0.0001) [p < 0.001; neonatal: 57.48%, p < 0.001) and Omicron-type SARS-CoV-2  with significance. The cord-to-maternal ratio was approximately 1.30 (IQR 0.78–2.23) and 0.71 (IQR 0.51–1.06) for the subjects that received two vaccine doses and those that received one dose, respectively. These data provide additional support for providing two vaccine doses for pregnant women and note that the Nab protection provided can be transferred to newborns. We found that this Nab protection was greatest when the interval between receiving the second vaccine dose and delivery was four-to eight-weeks, which is a finding supported by other articles as well [As with previous research , our stuine dose . Matsui  0.0001) . A high  0.0001) . Here, w as well ,37,38.As shown in From the previously published literature, the reduction and loss of vaccine protection may be related to weakened self-immunity or health status for antibody generation, different variants of SARS-CoV-2 that breach established immunity, diminished vaccine efficacy over time, and unimplemented physical barriers against virus infection. Additionally, the literature has shown that protection provided by a COVID-19 vaccine may be weakened six months after vaccination, and a breakthrough infection with a drop in antibody amount may be observed. COVID-19 booster vaccines have been found to enhance protection against SARS-CoV-2 and reduce the likelihood of subsequent severe illness ,40,41,42There were some limitations in our study. Because only 20 participants and 40 samples were used, the range and breadth of results in terms of clinical differences were limited. Additionally, because vaccine coverage is expanding in the population at large, it was difficult to find high numbers of pregnant women that were vaccinated after the onset of pregnancy, and the quality of the samples and lateral flow strip may affect both the examination and the calculation of the LOD. Other limitations included the fact that we only enrolled subjects that had received a particular vaccine, Moderna (mRNA-1273), and we only tested for protection against wild-type and Delta-type SARS-CoV-2. Additional variants may be studied in the future.In this study, we found that COVID-19 vaccine protection in pregnant women and neonates was better for wild-type virus compared to Delta-type and Omicron-type SARS-CoV-2 virus. Additionally, enrolled subjects that received two vaccine doses demonstrated better protection than those that received only one dose. We further found that a lateral flow immunoassay/spectrum analyzer approach could be used to detect the S1RBD IgG level cut-off value in maternal and neonatal serum, which was 60.15 and 156.31 ng/mL for Nab inhibition rates of at least 30% against Delta-type SARS-CoV-2. For those mothers with an S1RBD IgG level less than 60.15 ng/mL, an additional vaccine booster may be considered to augment low immunity/protection. For those neonates with an S1RBD IgG level less than 156.31 ng/mL, close protection with special care may be needed since there is no available vaccine for them. More cases may be needed in a future study for further clinical validation.